Therapeutic control of gastric ulcer disease depends upon interventions which control HCl secretion. The broad, long term objectives of this proposal are to define the molecules and mechanisms involved in regulated HCl secretion. HCl secretion involves the gastric H/K ATPase and a transport system for K to supply the H/K ATPase and for C1 to provide equivalents of C1 for HCl production. The working hypothesis to be tested in this proposal is that the apical membrane of the stimulated rabbit gastric parietal cell contains both C1 and K channels which are intimately associated with and essential for regulated gastric HCl secretion. The C1 channel, C1C-2G was cloned in this laboratory, and it is a rectifier which is controlled by Ca2+ or Mg2+, CaMKII as well as PKA, voltage, and extracellular pH. The K channel appears to be an inwardly rectifying K channel which is regulated by PKA and extracellular pH. Divalent cations may also play a role. A candidate K channel clone, RBHIK1, has been obtained and its properties will be compared and contrasted with those of the native channel. The physiologically relevant outward movement of both K and C1 are regulated by essentially the same effectors and respond similarly to changes in the electrical and chemical environments. The planned studies are directed toward understanding the interactions between intracellular ions in promoting rectification, the effects of protein kinases, voltage, and pH in promotion of outward movements of C1 by CIC-2G and of K by the native and cloned inwardly rectifying K channel. Functional studies will be complemented by correlated structural studies using channel cDNAs containing mutations of the cation binding sites, phosphorylation sites, and pH sensor sites. The Specific Aims which apply to both the CI and K channels are to study: 1) the role of cations in rectification of the channels; 2) the role of protein kinases in C1 and K channel function; 3) the role of protons in C1 and K channel function; and 4) the location of the channels in the gastric parietal cell in the non-secreting (resting) and secreting (stimulated) states. The first 3 aims will be studied using functional as well as correlated structural approaches. Aim 4 will be accomplished using structural probes based on knowledge of the sequence of the channels. These studies strive to address the fundamental questions regarding the molecules and mechanisms involved in regulated HCl secretion.